title: The identification and expression of the full-length HtrA2 gene from Penaeus monodon (black tiger shrimp).
abstract: HtrA2 is an apoptosis-activating protein to enhance the apoptotic process by preventing the formation of the IAP-caspase complex, thus freeing caspase to trigger the apoptosis pathway. Here, we presented the full-length sequence of HtrA2 from the black tiger shrimp (PmHtrA2). The full-length PmHtrA2 transcript was 1403 bp with a 1338 bp open reading frame encoding 445 amino acids and contains 5 conserved domains, namely, a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a serine protease domain, and a PDZ domain normally found in HtrA2 proteins of other organisms. The mature form of PmHtrA2 was cloned into the pET28b(+) and pET15bThio vectors, and the expression of the protein was compared in Escherichia coli BL21 DE3 and BL21 RIL (CodonPlus) strains. Greater quantities of stable and soluble PmHtrA2 were expressed as a thioredoxin fusion protein in E. coli BL21 RIL (CodonPlus) cells with the recombinant pET15bThio-PmHtrA2 vector. To investigate the expression of PmHtrA2 in shrimp, the crude proteins from several shrimp tissues were imaged by Western blot using the polyclonal antibody specific to the recombinant PmHtrA2. The expression of the 47-kDa immature PmHtrA2 protein could be detected in shrimp lysates from the gills and the muscles. This study is the first to report the full-length PmHtrA2 gene, which is functional in black tiger shrimp and will lead to more focused studies on the function of PmHtrA2 in apoptosis regulation during immune responses to viral infection in shrimp.
authors.affiliation: Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
authors.forename: Rukkit
authors.initials: R
authors.lastname: Suwannaboon
authors.forename: Kornsunee
authors.initials: K
authors.lastname: Phiwsaiya
authors.forename: Saengchan
authors.initials: S
authors.lastname: Senapin
authors.forename: Pongsak
authors.initials: P
authors.lastname: Khunrae
authors.forename: Triwit
authors.initials: T
authors.lastname: Rattanarojpong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins
chemical_list: D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D012697:Serine Endopeptidases
keywords: Apoptosis; Caspase; HtrA2; Penaeus monodon; Thioredoxin